Multiple sclerosis (MS) is a debilitating demyelinating disease of the central nervous system (CNS) that affects approximately 2.5 million people worldwide. US military personnel are at special risk to develop MS: the incidence rate in the US military population (12.9 per 100,000 person-years) is 1.7x higher than the civilian population, and 3x higher than the global population. Experimental autoimmune encephalomyelitis (EAE) is a widely studied animal model that shares many features of human MS. Tissue injury in EAE and MS is caused by inflammatory leukocytes that enter the CNS and destroy myelin. CNS-infiltrating, myelin-reactive CD4+ T cells play key roles in the pathology of MS. Although a number of MS treatments are available, due to the heterogeneity of the MS disease process, individual patient responses, and medication toxicities, there is a substantial unmet clinical need for improved therapeutics. T cell differentiation and function is profoundly affected by the engagement of metabolic pathways retinoid processing. Diacylglycerol O-acyltransferase-1 (DGAT1) is a metabolic enzyme that can catalyze the synthesis of triglycerides (TG, via DGAT activity), and retinyl esters (RE, via acyl CoA:retinol acyltransferase (ARAT) activity). Little is known regarding the role of DGAT1 in T cell biology. Our preliminary studies suggest that DGAT1 is selectively upregulated in activated mouse CD4+ T cells both in vitro and in vivo during EAE. DGAT1 is selectively expressed in brain lesions and CD4+ blood T cells obtained from MS patients. DGAT1 KO mice are protected against EAE, and DGAT1 pharmaco-inhibition suppresses EAE. Based on our preliminary data and the importance of retinoid metabolism in governing T cell differentiation and function, we hypothesize that T cell-expressed DGAT1 plays a key role in regulating pathogenic T cell activity in autoimmune demyelinating disease. In Aim 1 we will investigate the translational utility of targeting DGAT1 with small molecule antagonists to treat demyelinating disease. We will test the hypothesis that human blood CD4+ T cells express DGAT1 and contain DGAT/ARAT activity, and that DGAT1 regulates Treg and Th17 differentiation and function. We will also test the hypothesis that DGAT1 inhibitors administered after disease onset will reverse EAE progression and prevent relapse. Notably, small-molecule DGAT1 inhibitors are already being tested in clinical trials for treatment of obesity-associated diseases. Thus the studies in Aim 1 may uncover new MS-specific applications for existing drugs that have already cleared Phase I safety studies. In Aim 2 we propose to define the role of DGAT1 in CD4+ effector T cell formation and function. Using in vitro polarized CD4+ T cells, we will define DGAT1 expression and ARAT activity in mouse Treg and Th17 cells. Using naive T cells from WT and DGAT1 KO mice, we will define the role of DGAT1 in Treg and Th17 differentiation and function. To facilitate the study of DGAT1 in Treg development, we propose to generate DGAT1 KO/Foxp3/GFP mice (DGAT1 KO mice with GFP+ Tregs). In Aim 3, we propose to generate T cell conditional inducible DGAT1 KO mice (CD4CreERT2 -DGAT1fl/fl) to define the role of T cell-expressed DGAT1 in EAE. Finally, in Aim 4 we will investigate how dietary manipulation of vitamin A impacts the effects of DGAT1 deficiency in EAE. The results from this aim may provide a mechanistic rationale for vitamin A supplementation in MS, as low levels of vitamin A are associated with increased disease risk, and serum retinol levels are inversely correlated with magnetic resonance imaging outcomes in MS. Together, the proposed studies promise to elucidate novel insight into T cell immunometabolism and the role of DGAT1 in driving disease pathogenesis in MS, and holds great translational potential to reduce the impact of MS on US Veterans, their families, and the American public.